CD95 (APO-1/Fas) is a member of the death receptor family. Members of this family are characterized by the presence of the death domain (DD) in their cytoplasmic tails. Upon triggering, CD95 assembles a structure of molecules which we have called the "death inducing signaling complex" (DISC). The adapter molecule FADD/Mort1, a constituent molecule of the DISC, recruits the cysteine protease caspase-8, initiating the activation of a caspase cascade. Recent findings from our group have resulted in a model that the signaling cascade in CD95 mediated apoptosis occurs by two distinct pathways: mitochondria-independent and mitochondria-dependent. Cells which respond via these pathways are called Type I and Type II, respectively. Differences between these two apoptosis cell types can be found on many levels. First, the receptors appear to have different structures. Second, the formation of the DISC is severely impaired in Type II cells. As well, the two cell types vary in their sensitivity to apoptosis-inducing drugs that act through the mitochondria, such as C2 ceramide. Finally, caspase-8 acts as an initiator caspase in Type I cells, while it functions primarily as an effector caspase in Type II cells. These differences provide the basis for the differential modulation of the apoptosis sensitivity of these cell types. One goal of this study is to test our model in normal cells and to test the response of these cells to different forms of CD95 ligand (L) (soluble and membrane bound CD95L). We also plan to characterize the mechanism that activates mitochondria associated caspase-8 in Type Il cells. Another major question is whether cells are restricted to one cell type or whether they can switch during activation or differentiation. Overall, our goal is to further understand the triggers and the regulation of each pathway. To do this, we have three specific aims: Specific Aim number 1: Determination of the different requirements to induce CD95 mediated apoptosis in Type I and Type II cells at the receptor level. Specific Aim number 2: Characterization of the mitochondrial branch of the CD95 Type II pathway. Specific Aim number 3: Identification of the mechanisms that regulate differentiation from one cell type to another. The completion of this work will result in a much better understanding of the apoptotic process in general and should ultimately lead to new strategies for intervention in the many diseases where dysregulation of death receptor mediated apoptosis plays a central role.